Bottom Line:
Salmonella enterica can cause intestinal or systemic infections in humans and animals mainly by the presence of pathogenicity islands SPI-1 and SPI-2, containing 39 and 44 genes, respectively.The AraC-like regulator HilD positively controls the expression of the SPI-1 genes, as well as many other Salmonella virulence genes including those located in SPI-2.Additionally, we show that in the absence of the Lon protease, which degrades HilD, the CpxR-P-mediated repression of the SPI-1 genes is mostly lost; moreover, we demonstrate that CpxR-P negatively affects the stability of HilD and thus decreases the expression of HilD-target genes, such as hilD itself and hilA, located in SPI-1.

ABSTRACTSalmonella enterica can cause intestinal or systemic infections in humans and animals mainly by the presence of pathogenicity islands SPI-1 and SPI-2, containing 39 and 44 genes, respectively. The AraC-like regulator HilD positively controls the expression of the SPI-1 genes, as well as many other Salmonella virulence genes including those located in SPI-2. A previous report indicates that the two-component system CpxR/A regulates the SPI-1 genes: the absence of the sensor kinase CpxA, but not the absence of its cognate response regulator CpxR, reduces their expression. The presence and absence of cell envelope stress activates kinase and phosphatase activities of CpxA, respectively, which in turn controls the level of phosphorylated CpxR (CpxR-P). In this work, we further define the mechanism for the CpxR/A-mediated regulation of SPI-1 genes. The negative effect exerted by the absence of CpxA on the expression of SPI-1 genes was counteracted by the absence of CpxR or by the absence of the two enzymes, AckA and Pta, which render acetyl-phosphate that phosphorylates CpxR. Furthermore, overexpression of the lipoprotein NlpE, which activates CpxA kinase activity on CpxR, or overexpression of CpxR, repressed the expression of SPI-1 genes. Thus, our results provide several lines of evidence strongly supporting that the absence of CpxA leads to the phosphorylation of CpxR via the AckA/Pta enzymes, which represses both the SPI-1 and SPI-2 genes. Additionally, we show that in the absence of the Lon protease, which degrades HilD, the CpxR-P-mediated repression of the SPI-1 genes is mostly lost; moreover, we demonstrate that CpxR-P negatively affects the stability of HilD and thus decreases the expression of HilD-target genes, such as hilD itself and hilA, located in SPI-1. Our data further expand the insight on the different regulatory pathways for gene expression involving CpxR/A and on the complex regulatory network governing virulence in Salmonella.

Figure 4: CpxR represses the autoregulation of hilD and thus negatively affects the expression of hilA. Expression of the hilD-cat transcriptional fusion carried by plasmid philD-cat was tested in the WT S. Typhimurium strain carrying plasmid pK3-CpxR or pK3-RpoH, or the vector pMPM-K3 (A), as well as in the WT S. Typhimurium strain and its isogenic ΔhilD, ΔcpxA, and ΔhilD ΔcpxA mutants (B). Expression of the hilA-cat transcriptional fusion carried by plasmid philA-cat was tested in the WT S. Typhimurium strain (C), or in its isogenic ΔhilD ΔCthns mutant (D), containing plasmid pK3-CpxR or pK3-RpoH, or the vector pMPM-K3. Plasmids pK3-CpxR and pK3-RpoH, as well as Salmonella, lack the gene encoding the repressor LacI and thus they constitutively express CpxR and RpoH, respectively, from a lac promoter. CAT-specific activity was determined from samples collected of bacterial cultures grown for 5 h in LB medium at 37°C. The data are the averages of three different experiments performed in duplicate. Bars represent the standard deviations. *Expression statistically different with respect to that shown by the same fusion in the WT strain containing the vector. **Expression statistically different with respect to that shown by the same fusion in the WT strain.

Mentions:
Several global regulators control the expression of the SPI-1 genes by directly affecting the expression, activity or concentration of HilD or HilA, the central regulators of these genes (Golubeva et al., 2012; Fàbrega and Vila, 2013). Our results indicate that CpxR represses the expression of HilA (Figure 1C). To start to define whether CpxR affects hilA directly or through HilD, which positively regulates hilA, we determined the effect of the overexpression of CpxR on the activity of a hilD-cat transcriptional fusion in the WT S. Typhimurium strain. Since plasmid pCA-CpxR, expressing the E. coli K12 CpxR, is incompatible with the vector carrying the cat fusions tested, for the next assays we constructed and used the plasmid pK3-CpxR, which constitutively expresses CpxR of S. Typhimurium 14028s. The overexpression of CpxR reduced 50% the expression of the hilD-cat fusion (Figure 4A), revealing that CpxR represses hilD. CpxR could directly repress the transcription of hilD or reduce post-transcriptionally the concentration of HilD and thus affect its positive autoregulation. To determine if CpxR affects the autoregulation of hilD, the expression of the hilD-cat fusion was determined in the WT S. Typhimurium strain and its derivatives ΔcpxA, ΔhilD, and ΔhilD ΔcpxA mutants. As shown in Figure 4B, the expression of the hilD-cat fusion was similarly reduced in these three mutants, indicating that the absence of CpxA or HilD has the same effect on the expression of hilD, and that, when HilD is not present, the absence of CpxA does not longer repress hilD. In contrast, the expression of the cpxRA-cat fusion was similarly increased in the ΔcpxA and ΔhilD ΔcpxA mutants (Figure 1D), indicating that the absence of CpxA activates the expression of cpxRA independently of HilD. These results suggest that CpxR represses hilA and thus the other SPI-1 genes by affecting the autoregulation of HilD. To confirm that CpxR regulates hilA through HilD and not directly, we analyzed the effect of CpxR on the expression of hilA in the presence or not of HilD. Previous studies indicate that HilD induces the expression of hilA by counteracting the repression exerted by the nucleoid protein H-NS on the promoter of this gene (Schechter et al., 1999; Schechter and Lee, 2001; Olekhnovich and Kadner, 2006); thus, in the absence of H-NS activity hilA can be expressed independently of HilD. Full-length deletion of hns produces severe growth defects in S. Typhimurium (Lucchini et al., 2006; Navarre et al., 2006). However, deletion of the sequence encoding the C-terminal region of H-NS (ΔCthns), which contains its DNA-binding domain, has only a minor effect on S. Typhimurium fitness (Fernández-Mora, personal communication), probably because the N-terminal of H-NS can still repress some of its target genes by interacting with StpA, another nucleoid protein (Free et al., 2001). Therefore, we constructed and tested a S. Typhimurium 14028s ΔhilD ΔCthns mutant. The overexpression of CpxR, from plasmid pK3-CpxR, reduced five-fold the expression of a hilA-cat transcriptional fusion in the WT strain (Figure 4C), but did not affect the high levels of expression showed by this fusion in the ΔhilD ΔCthns mutant (Figure 4D), indicating that CpxR regulates hilA through HilD and not directly.

Figure 4: CpxR represses the autoregulation of hilD and thus negatively affects the expression of hilA. Expression of the hilD-cat transcriptional fusion carried by plasmid philD-cat was tested in the WT S. Typhimurium strain carrying plasmid pK3-CpxR or pK3-RpoH, or the vector pMPM-K3 (A), as well as in the WT S. Typhimurium strain and its isogenic ΔhilD, ΔcpxA, and ΔhilD ΔcpxA mutants (B). Expression of the hilA-cat transcriptional fusion carried by plasmid philA-cat was tested in the WT S. Typhimurium strain (C), or in its isogenic ΔhilD ΔCthns mutant (D), containing plasmid pK3-CpxR or pK3-RpoH, or the vector pMPM-K3. Plasmids pK3-CpxR and pK3-RpoH, as well as Salmonella, lack the gene encoding the repressor LacI and thus they constitutively express CpxR and RpoH, respectively, from a lac promoter. CAT-specific activity was determined from samples collected of bacterial cultures grown for 5 h in LB medium at 37°C. The data are the averages of three different experiments performed in duplicate. Bars represent the standard deviations. *Expression statistically different with respect to that shown by the same fusion in the WT strain containing the vector. **Expression statistically different with respect to that shown by the same fusion in the WT strain.

Mentions:
Several global regulators control the expression of the SPI-1 genes by directly affecting the expression, activity or concentration of HilD or HilA, the central regulators of these genes (Golubeva et al., 2012; Fàbrega and Vila, 2013). Our results indicate that CpxR represses the expression of HilA (Figure 1C). To start to define whether CpxR affects hilA directly or through HilD, which positively regulates hilA, we determined the effect of the overexpression of CpxR on the activity of a hilD-cat transcriptional fusion in the WT S. Typhimurium strain. Since plasmid pCA-CpxR, expressing the E. coli K12 CpxR, is incompatible with the vector carrying the cat fusions tested, for the next assays we constructed and used the plasmid pK3-CpxR, which constitutively expresses CpxR of S. Typhimurium 14028s. The overexpression of CpxR reduced 50% the expression of the hilD-cat fusion (Figure 4A), revealing that CpxR represses hilD. CpxR could directly repress the transcription of hilD or reduce post-transcriptionally the concentration of HilD and thus affect its positive autoregulation. To determine if CpxR affects the autoregulation of hilD, the expression of the hilD-cat fusion was determined in the WT S. Typhimurium strain and its derivatives ΔcpxA, ΔhilD, and ΔhilD ΔcpxA mutants. As shown in Figure 4B, the expression of the hilD-cat fusion was similarly reduced in these three mutants, indicating that the absence of CpxA or HilD has the same effect on the expression of hilD, and that, when HilD is not present, the absence of CpxA does not longer repress hilD. In contrast, the expression of the cpxRA-cat fusion was similarly increased in the ΔcpxA and ΔhilD ΔcpxA mutants (Figure 1D), indicating that the absence of CpxA activates the expression of cpxRA independently of HilD. These results suggest that CpxR represses hilA and thus the other SPI-1 genes by affecting the autoregulation of HilD. To confirm that CpxR regulates hilA through HilD and not directly, we analyzed the effect of CpxR on the expression of hilA in the presence or not of HilD. Previous studies indicate that HilD induces the expression of hilA by counteracting the repression exerted by the nucleoid protein H-NS on the promoter of this gene (Schechter et al., 1999; Schechter and Lee, 2001; Olekhnovich and Kadner, 2006); thus, in the absence of H-NS activity hilA can be expressed independently of HilD. Full-length deletion of hns produces severe growth defects in S. Typhimurium (Lucchini et al., 2006; Navarre et al., 2006). However, deletion of the sequence encoding the C-terminal region of H-NS (ΔCthns), which contains its DNA-binding domain, has only a minor effect on S. Typhimurium fitness (Fernández-Mora, personal communication), probably because the N-terminal of H-NS can still repress some of its target genes by interacting with StpA, another nucleoid protein (Free et al., 2001). Therefore, we constructed and tested a S. Typhimurium 14028s ΔhilD ΔCthns mutant. The overexpression of CpxR, from plasmid pK3-CpxR, reduced five-fold the expression of a hilA-cat transcriptional fusion in the WT strain (Figure 4C), but did not affect the high levels of expression showed by this fusion in the ΔhilD ΔCthns mutant (Figure 4D), indicating that CpxR regulates hilA through HilD and not directly.

Bottom Line:
Salmonella enterica can cause intestinal or systemic infections in humans and animals mainly by the presence of pathogenicity islands SPI-1 and SPI-2, containing 39 and 44 genes, respectively.The AraC-like regulator HilD positively controls the expression of the SPI-1 genes, as well as many other Salmonella virulence genes including those located in SPI-2.Additionally, we show that in the absence of the Lon protease, which degrades HilD, the CpxR-P-mediated repression of the SPI-1 genes is mostly lost; moreover, we demonstrate that CpxR-P negatively affects the stability of HilD and thus decreases the expression of HilD-target genes, such as hilD itself and hilA, located in SPI-1.

ABSTRACTSalmonella enterica can cause intestinal or systemic infections in humans and animals mainly by the presence of pathogenicity islands SPI-1 and SPI-2, containing 39 and 44 genes, respectively. The AraC-like regulator HilD positively controls the expression of the SPI-1 genes, as well as many other Salmonella virulence genes including those located in SPI-2. A previous report indicates that the two-component system CpxR/A regulates the SPI-1 genes: the absence of the sensor kinase CpxA, but not the absence of its cognate response regulator CpxR, reduces their expression. The presence and absence of cell envelope stress activates kinase and phosphatase activities of CpxA, respectively, which in turn controls the level of phosphorylated CpxR (CpxR-P). In this work, we further define the mechanism for the CpxR/A-mediated regulation of SPI-1 genes. The negative effect exerted by the absence of CpxA on the expression of SPI-1 genes was counteracted by the absence of CpxR or by the absence of the two enzymes, AckA and Pta, which render acetyl-phosphate that phosphorylates CpxR. Furthermore, overexpression of the lipoprotein NlpE, which activates CpxA kinase activity on CpxR, or overexpression of CpxR, repressed the expression of SPI-1 genes. Thus, our results provide several lines of evidence strongly supporting that the absence of CpxA leads to the phosphorylation of CpxR via the AckA/Pta enzymes, which represses both the SPI-1 and SPI-2 genes. Additionally, we show that in the absence of the Lon protease, which degrades HilD, the CpxR-P-mediated repression of the SPI-1 genes is mostly lost; moreover, we demonstrate that CpxR-P negatively affects the stability of HilD and thus decreases the expression of HilD-target genes, such as hilD itself and hilA, located in SPI-1. Our data further expand the insight on the different regulatory pathways for gene expression involving CpxR/A and on the complex regulatory network governing virulence in Salmonella.